Acinetobacter baumannii is a Gram-negative bacterium and emerging human pathogen. A. baumannii is highly prevalent and increasingly important as a nosocomial infectious agent, as these bacteria have the ability to effectively colonize metal and plastic surfaces common in healthcare-associated settings. Type IV Pili (T4P) are hair-like surface appendages produced by many species of pathogenic Gram negative bacteria which play a role in diverse processes such as cellular adhesion, colonization, twitching motility, biofilm formation, horizontal gene transfer and in numerous instances are essential for virulence. T4Ps are composed exclusively or primarily of many copies of pilin protein, tightly packed in a helix so that the highly hydrophobic amino-terminus of the pilin is buried in the pilus core. In A. baumanii, type IV pili have been found by our group and others, to be essential for twitching motility and horizontal gene transfer and to promote both biofilm formation and adhesion to host cells. Recent results from our group indicate that type IV pili in Acinetobacter are essential for both twitching motility and biofilm formation and that pilins from A. baumannii strains ACICU, AB5075 and BIDMC75 are structurally divergent. By complementing a tipilA mutant with pilA genes from these distinct T4P subtypes, our group is the first to demonstrate that differences in pilA are sufficient to reproduce differences in twitching motility and biofilm formation. The objective of this application is to determine the molecular basis for the observed functional specialization in Acinetobacter type IV pili. My central hypothesis is that the biases between biofilm formation and surface motility are the product of differences in the equilibrium between individual cells and microcolonies of cells attached through intertwined bundles of type IV pili. To evaluate the molecular basis for this evolutionary divergence, we will i) measure the equilibrium between individual and bundled pili in divergent Acinetobacter T4P subtypes and ii) Quantify the relationship between Acinetobacter microcolony formation and twitching motility.